Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and ... more Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and while most of the 4,000 new cases of pediatric ITP diagnosed each year self-resolve, 10% of these patients have major bleeding episodes. The platelet count remains the mainstay method for predicting hemorrhage and unfortunately has only proven to be loosely correlated to bleeding severity. Consequently, there currently exists no biomarker that accurately and reliably predicts which patients need immediate medical treatment, all of which have side effects, and which patients only require monitoring. To that end, we leveraged our Platelet Contraction Cytometer (PCC), a versatile system that measures platelet contraction forces at the single-cell level and at high-throughput, to study platelets of patients with ITP prospectively. Buchanan bleeding scores were used to distinguish patients with severe symptoms from asymptomatic patients. With 49 patients, we observed two significant findings: 1) Tracking both single platelet force measurements and platelet count enables stratifying patients into having major, minor, or no risk for bleeding. Accordingly, patients in the major risk category have a combined low platelet force and low platelet count. 2) Longitudinal studies showed that when major risk patients had increased platelet force or higher platelet counts, this was associated with the alleviation of major symptoms. Thus, when utilized together, platelet force and platelet count can more accurately predict bleeding severity in pediatric ITP patients. Platelet Contraction Cytometry: Our PCC utilizes a large array of fibrinogen "microdot" pairs patterned on a flat hydrogel of known stiffness. Thrombin-activated platelets adhere to these microdots, spread to the neighboring microdot, and contract, pulling the microdots closer together. As platelet force is directly proportional to the microdot displacement, only a single microscopy measurement is necessary to determine the force applied by each single platelet (Fig 1). As such, hundreds of platelets are measured in a controlled mechanical and biochemical environment (Myers et al, 2017) Results: With our cohort of 49 ITP patients, we observed that low average single cell platelet contraction forces highly correlate with severe bleeding symptoms (Buchannan scores 2-4). Using a regression tree, we found that an average force of 26nN best separates symptomatic from asymptomatic patients with a diagnostic sensitivity of 100% and specificity of 86.8% (AUC 0.97, 95% CI: 0.9361-1). However, platelets from some asymptomatic patients exerted forces of less than 26nN as well. In a more thorough examination, we found that majority of those asymptomatic patients with low platelet forces had platelet counts > 40,000/uL. Although our data shows that platelet count alone is a poor predictor of severe bleeding symptoms (AUC 0.8, 95% CI: 0.6757-0.9189), when a platelet count <40k/uL is coupled alongside our criterion of average contraction force <26 nN, we find that the specificity of our system increases to 94.7% and thus platelet count and platelet force synergistically predict which patients are symptomatic with high accuracy. Using a logistic regression model, we also found that with each 1nN decrease in average contraction force, the odds of the patient bleeding increases 2.1 (95% CI: 1.1-4.0) times that of being asymptomatic and when this decrease in contraction force is increased 5nN, the odds of a patient bleeding is 44.4 (95% CI: 1.8-1066.5) times that of being asymptomatic (Fig 2A-B). Moreover, when tracking individual patients (n=4) over time, we demonstrate that whenever a patient's blood sample is associated with increased single platelet average contraction force >26 nN or platelet count >40k/uL, bleeding symptoms are alleviated. Conversely, whenever a patient exhibited high platelet contractile force and low platelet count, the onset of bleeding symptoms correlated with a decrease in platelet forces. Unlike bulk or micro-clot assays, our assay functions as a "stress test" of single platelets by placing them in conditions that maximize contractility, and in both cases, the reduction in the subpopulation of highly contractile platelets is correlated with bleeding severity. As such, our work suggests that single platelet forces could be used as a diagnostic biomarker to assess bleeding risk in patients with ITP regardless of count (Fig 2C-D). Disclosures Bennett: Novartis: Research Funding. Lam:Sanguina, LLC: Equity Ownership.
One fifth of school aged children in the US have a chronic medical illness and represent an under... more One fifth of school aged children in the US have a chronic medical illness and represent an underserved and educationally disadvantaged population. In particular, children with sickle cell disease (SCD) of all genotypes spend significant amounts of time in the medical settings and represent a potentially "captive audience" where hospitalization time can include educational activities. Accordingly, we saw an opportunity for Biomedical Engineering (BME) undergraduates at Georgia Institute of Technology, who are taught biology, medicine, engineering, and design to implement a new paradigm of K-12 Science, Technology, Engineering, and Mathematics (STEM) programming, where human physiology is used as a model framework. In collaboration with hospital school teachers at Children's Healthcare of Atlanta, we created a novel educational program where we (1) leverage the patient's own medical experience as motivation for learning, (2) develop hands-on interactive activities to teach STEM concepts that are adaptable for the patient's cognitive level, (3) provide undergraduate students with high quality meaningful patient interactions and clinical experiences, and (4) provide innovative educational programming for hospitals. The BME undergraduates enrolled in a human-centered design course focusing on iterative design, development and implementation of hands-on interactive STEM activities rooted in human physiology (Fig. 1A). Each activity contains state and national K-12 STEM standards. The BME undergraduates are well-suited to teach in the hospital environment, as they experience a strong STEM curriculum and are able to capture the true interdisciplinary nature of medical science in each activity. The undergraduates and patients are near-peer age, relationship-building occurs quickly, the patients admire them and enjoy their time together. During the 2018-19 school year we developed learning assessment questions for 2 of the most requested activities, assessing each SCD patient's mastery of the learning objectives. In the Blood Jar activity (Fig. 1B), the SCD patient builds a model of blood, learning the composition, function of each component, and importance of hydration. Sixty-five patients participated, 43% of grade K - 2, 61% of 3 - 5, 85% of 6th through High School scored Thoroughly Demonstrated. In the Bone activity (Fig. 1C), each SCD patient constructs a bone model to understand the function and structure of a bone. Twenty-nine patients participated, 28% of grade K - 2, 30% of 3 - 5, 88% of 6 - 8, and 75% of high school scored Thoroughly Demonstrated. Patient feedback (Fig. 1D) has been overwhelmingly positive, stating the activities are "fun" and "engaging" and requesting additional visits from the BME undergraduates. BME undergraduate expressed growth and learning of: disease pathophysiology, teaching and learning styles, and effective and succinct communication skills. In Georgia, Life Science is taught in 5th and 7th grade, our target demographic. Our results show at least 75% of patients in 6th grade and above demonstrated thorough understanding of the STEM topics taught, while K - 5 grade did not achieve that level of understanding. Lower scores for those patients are attributed to difficulties remembering new scientific vocabulary. Many of the BME undergraduates in our program are pre-med and use the experiences and meaningful interactions with patients in their medical school applications. As stated by a BME undergraduate after a patient interaction, "the white blood cells are like soldiers in the army! I like that! This analogy prompted the patient to think of his future. It reminded me the littlest things can make a big difference. This makes me even more excited to go into medicine." Our program represents an innovative approach to teaching STEM by: engaging SCD children, who are uniquely suited to learn important STEM concepts within the context of their own disease, provide learning to SCD patients during extended absences from school, allowing BME undergraduate students to design, develop, and teach STEM activities, provide meaningful clinical experience, and supplying quality educational programming for the hospital. This collaborative program can be straightforwardly implemented at other pediatric institutions with robust college volunteer programs and hospital school programs. Our activities can be exported and implemented at other institutions. Disclosures Lam: Sanguina, Inc: Current equity holder in private company.
Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and ... more Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and while most of the 4,000 new cases of pediatric ITP diagnosed each year self-resolve, 10% of these patients have major bleeding episodes. The platelet count remains the mainstay method for predicting hemorrhage and unfortunately has only proven to be loosely correlated to bleeding severity. Consequently, there currently exists no biomarker that accurately and reliably predicts which patients need immediate medical treatment, all of which have side effects, and which patients only require monitoring. To that end, we leveraged our Platelet Contraction Cytometer (PCC), a versatile system that measures platelet contraction forces at the single-cell level and at high-throughput, to study platelets of patients with ITP prospectively. Buchanan bleeding scores were used to distinguish patients with severe symptoms from asymptomatic patients. With 49 patients, we observed two significant findings: 1) Tracking both single platelet force measurements and platelet count enables stratifying patients into having major, minor, or no risk for bleeding. Accordingly, patients in the major risk category have a combined low platelet force and low platelet count. 2) Longitudinal studies showed that when major risk patients had increased platelet force or higher platelet counts, this was associated with the alleviation of major symptoms. Thus, when utilized together, platelet force and platelet count can more accurately predict bleeding severity in pediatric ITP patients. Platelet Contraction Cytometry: Our PCC utilizes a large array of fibrinogen "microdot" pairs patterned on a flat hydrogel of known stiffness. Thrombin-activated platelets adhere to these microdots, spread to the neighboring microdot, and contract, pulling the microdots closer together. As platelet force is directly proportional to the microdot displacement, only a single microscopy measurement is necessary to determine the force applied by each single platelet (Fig 1). As such, hundreds of platelets are measured in a controlled mechanical and biochemical environment (Myers et al, 2017) Results: With our cohort of 49 ITP patients, we observed that low average single cell platelet contraction forces highly correlate with severe bleeding symptoms (Buchannan scores 2-4). Using a regression tree, we found that an average force of 26nN best separates symptomatic from asymptomatic patients with a diagnostic sensitivity of 100% and specificity of 86.8% (AUC 0.97, 95% CI: 0.9361-1). However, platelets from some asymptomatic patients exerted forces of less than 26nN as well. In a more thorough examination, we found that majority of those asymptomatic patients with low platelet forces had platelet counts > 40,000/uL. Although our data shows that platelet count alone is a poor predictor of severe bleeding symptoms (AUC 0.8, 95% CI: 0.6757-0.9189), when a platelet count <40k/uL is coupled alongside our criterion of average contraction force <26 nN, we find that the specificity of our system increases to 94.7% and thus platelet count and platelet force synergistically predict which patients are symptomatic with high accuracy. Using a logistic regression model, we also found that with each 1nN decrease in average contraction force, the odds of the patient bleeding increases 2.1 (95% CI: 1.1-4.0) times that of being asymptomatic and when this decrease in contraction force is increased 5nN, the odds of a patient bleeding is 44.4 (95% CI: 1.8-1066.5) times that of being asymptomatic (Fig 2A-B). Moreover, when tracking individual patients (n=4) over time, we demonstrate that whenever a patient's blood sample is associated with increased single platelet average contraction force >26 nN or platelet count >40k/uL, bleeding symptoms are alleviated. Conversely, whenever a patient exhibited high platelet contractile force and low platelet count, the onset of bleeding symptoms correlated with a decrease in platelet forces. Unlike bulk or micro-clot assays, our assay functions as a "stress test" of single platelets by placing them in conditions that maximize contractility, and in both cases, the reduction in the subpopulation of highly contractile platelets is correlated with bleeding severity. As such, our work suggests that single platelet forces could be used as a diagnostic biomarker to assess bleeding risk in patients with ITP regardless of count (Fig 2C-D). Disclosures Bennett: Novartis: Research Funding. Lam:Sanguina, LLC: Equity Ownership.
One fifth of school aged children in the US have a chronic medical illness and represent an under... more One fifth of school aged children in the US have a chronic medical illness and represent an underserved and educationally disadvantaged population. In particular, children with sickle cell disease (SCD) of all genotypes spend significant amounts of time in the medical settings and represent a potentially "captive audience" where hospitalization time can include educational activities. Accordingly, we saw an opportunity for Biomedical Engineering (BME) undergraduates at Georgia Institute of Technology, who are taught biology, medicine, engineering, and design to implement a new paradigm of K-12 Science, Technology, Engineering, and Mathematics (STEM) programming, where human physiology is used as a model framework. In collaboration with hospital school teachers at Children's Healthcare of Atlanta, we created a novel educational program where we (1) leverage the patient's own medical experience as motivation for learning, (2) develop hands-on interactive activities to teach STEM concepts that are adaptable for the patient's cognitive level, (3) provide undergraduate students with high quality meaningful patient interactions and clinical experiences, and (4) provide innovative educational programming for hospitals. The BME undergraduates enrolled in a human-centered design course focusing on iterative design, development and implementation of hands-on interactive STEM activities rooted in human physiology (Fig. 1A). Each activity contains state and national K-12 STEM standards. The BME undergraduates are well-suited to teach in the hospital environment, as they experience a strong STEM curriculum and are able to capture the true interdisciplinary nature of medical science in each activity. The undergraduates and patients are near-peer age, relationship-building occurs quickly, the patients admire them and enjoy their time together. During the 2018-19 school year we developed learning assessment questions for 2 of the most requested activities, assessing each SCD patient's mastery of the learning objectives. In the Blood Jar activity (Fig. 1B), the SCD patient builds a model of blood, learning the composition, function of each component, and importance of hydration. Sixty-five patients participated, 43% of grade K - 2, 61% of 3 - 5, 85% of 6th through High School scored Thoroughly Demonstrated. In the Bone activity (Fig. 1C), each SCD patient constructs a bone model to understand the function and structure of a bone. Twenty-nine patients participated, 28% of grade K - 2, 30% of 3 - 5, 88% of 6 - 8, and 75% of high school scored Thoroughly Demonstrated. Patient feedback (Fig. 1D) has been overwhelmingly positive, stating the activities are "fun" and "engaging" and requesting additional visits from the BME undergraduates. BME undergraduate expressed growth and learning of: disease pathophysiology, teaching and learning styles, and effective and succinct communication skills. In Georgia, Life Science is taught in 5th and 7th grade, our target demographic. Our results show at least 75% of patients in 6th grade and above demonstrated thorough understanding of the STEM topics taught, while K - 5 grade did not achieve that level of understanding. Lower scores for those patients are attributed to difficulties remembering new scientific vocabulary. Many of the BME undergraduates in our program are pre-med and use the experiences and meaningful interactions with patients in their medical school applications. As stated by a BME undergraduate after a patient interaction, "the white blood cells are like soldiers in the army! I like that! This analogy prompted the patient to think of his future. It reminded me the littlest things can make a big difference. This makes me even more excited to go into medicine." Our program represents an innovative approach to teaching STEM by: engaging SCD children, who are uniquely suited to learn important STEM concepts within the context of their own disease, provide learning to SCD patients during extended absences from school, allowing BME undergraduate students to design, develop, and teach STEM activities, provide meaningful clinical experience, and supplying quality educational programming for the hospital. This collaborative program can be straightforwardly implemented at other pediatric institutions with robust college volunteer programs and hospital school programs. Our activities can be exported and implemented at other institutions. Disclosures Lam: Sanguina, Inc: Current equity holder in private company.
Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absen... more Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absence of any known causes and affects over 4,000 US children and 8,000 adults each year (Terrell et al, 2010). Deciding when and how to treat these patients remains difficult as there is no biomarker or diagnostic test that identifies which patients will self-resolve and which are at risk for major bleeding and life-threatening intracranial hemorrhage (~10%). In addition, the medications used to treat ITP all have significant side effects, forcing clinicians to balance risks associated with bleeding and medication. As such, an ongoing debate in the field of clinical hematology centers around which patients require therapy (Flores & Buchanan, 2013)(Cooper 2017)(Gralnek 2008). Here, we show that a new quantitative measurement of platelet function, which is independent of known platelet biomarkers (Myers et al, 2017), has diagnostic potential in identifying bleeding risk in ITP patients. Specifically, using high-throughput platelet contraction cytometry (PCC) measurements of individual platelet forces (Myers et al, 2017), we found that low platelet forces strongly correlate with bleeding symptoms. Unlike existing tests of platelet function that use bulk measurements, our test operates at the single cell level and therefore does not depend on the platelet count, enabling a direct comparison of individual platelet function in health versus disease states. In the broad context of overall function, our findings of impaired force generation agree with previous research demonstrating that impaired platelet function, and not low platelet count, correlates with bleeding in ITP patients (Frelinger et al, 2015). In addition, as the first demonstration of a correlation between single cell force and disease, our novel approach may represent an entirely new class of "physics-based" diagnostics. Platelet Contraction Cytometry (PCC): Within the PCC, a single platelet attaches, spreads, and applies contractile force to a pair of fibrinogen microdots that are attached to a moveable, spring-like, surface. Since the applied platelet contractile force is directly proportional to the microdot displacement, the force is calculated from a single fluorescence image of the platelet. Using microfabrication technology, thousands of microdots are created on a single device to enable high-throughput measurements in tightly controlled mechanical, biochemical, and shear microenvironments (Fig 1). Here, gel-filtered platelets from patients are activated (1U/mL thrombin), plated on the device with a moderately stiff surface (75 kPa), and measured as described previously (Myers et al, 2017). Results: In a cohort of ITP patients (n = 27), we observed that patients with bleeding and/or bruising symptoms exhibited significantly lower average platelet contraction forces than asymptomatic patients regardless of platelet count. Using an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity. From a mechanistic perspective, our preliminary data also suggests that mean platelet volume does not correlate with platelet force or function, although further studies on a single platelet level are needed to confirm this result. While this data indicates that patients with bleeding symptoms have platelets with low forces, it is unclear whether low forces correlate with bleeding or whether the patient always has had low platelet forces that therefore render them susceptible to bleeding. Our preliminary prospective data suggests that low platelet forces correlate with the bleeding symptoms themselves and return to higher values when bleeding symptoms cease. Importantly, as our approach enables "single-cell" examinations of platelet forces, we identified different platelet subpopulations unique to ITP. Healthy individuals have a distribution of contractile forces with a single peak, while ITP patients tend to have two prominent peaks, one with a lower contractile force and one with a higher one. Surprisingly, patients with bleeding symptoms only have a low force peak, suggesting that the lack of highly contractile platelets may be a biophysical biomarker for bleeding (Fig 2). Ongoing studies are focused on using this finding to further improve specificity as well as elucidating the mechanistic underpinnings of low platelet contraction in ITP. Disclosures No relevant conflicts of interest to declare.
Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absen... more Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absence of any known causes and affects over 4,000 US children and 8,000 adults each year (Terrell et al, 2010). Deciding when and how to treat these patients remains difficult as there is no biomarker or diagnostic test that identifies which patients will self-resolve and which are at risk for major bleeding and life-threatening intracranial hemorrhage (~10%). In addition, the medications used to treat ITP all have significant side effects, forcing clinicians to balance risks associated with bleeding and medication. As such, an ongoing debate in the field of clinical hematology centers around which patients require therapy (Flores & Buchanan, 2013)(Cooper 2017)(Gralnek 2008). Here, we show that a new quantitative measurement of platelet function, which is independent of known platelet biomarkers (Myers et al, 2017), has diagnostic potential in identifying bleeding risk in ITP patients. Specifically, using high-throughput platelet contraction cytometry (PCC) measurements of individual platelet forces (Myers et al, 2017), we found that low platelet forces strongly correlate with bleeding symptoms. Unlike existing tests of platelet function that use bulk measurements, our test operates at the single cell level and therefore does not depend on the platelet count, enabling a direct comparison of individual platelet function in health versus disease states. In the broad context of overall function, our findings of impaired force generation agree with previous research demonstrating that impaired platelet function, and not low platelet count, correlates with bleeding in ITP patients (Frelinger et al, 2015). In addition, as the first demonstration of a correlation between single cell force and disease, our novel approach may represent an entirely new class of "physics-based" diagnostics. Platelet Contraction Cytometry (PCC): Within the PCC, a single platelet attaches, spreads, and applies contractile force to a pair of fibrinogen microdots that are attached to a moveable, spring-like, surface. Since the applied platelet contractile force is directly proportional to the microdot displacement, the force is calculated from a single fluorescence image of the platelet. Using microfabrication technology, thousands of microdots are created on a single device to enable high-throughput measurements in tightly controlled mechanical, biochemical, and shear microenvironments (Fig 1). Here, gel-filtered platelets from patients are activated (1U/mL thrombin), plated on the device with a moderately stiff surface (75 kPa), and measured as described previously (Myers et al, 2017). Results: In a cohort of ITP patients (n = 27), we observed that patients with bleeding and/or bruising symptoms exhibited significantly lower average platelet contraction forces than asymptomatic patients regardless of platelet count. Using an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity. From a mechanistic perspective, our preliminary data also suggests that mean platelet volume does not correlate with platelet force or function, although further studies on a single platelet level are needed to confirm this result. While this data indicates that patients with bleeding symptoms have platelets with low forces, it is unclear whether low forces correlate with bleeding or whether the patient always has had low platelet forces that therefore render them susceptible to bleeding. Our preliminary prospective data suggests that low platelet forces correlate with the bleeding symptoms themselves and return to higher values when bleeding symptoms cease. Importantly, as our approach enables "single-cell" examinations of platelet forces, we identified different platelet subpopulations unique to ITP. Healthy individuals have a distribution of contractile forces with a single peak, while ITP patients tend to have two prominent peaks, one with a lower contractile force and one with a higher one. Surprisingly, patients with bleeding symptoms only have a low force peak, suggesting that the lack of highly contractile platelets may be a biophysical biomarker for bleeding (Fig 2). Ongoing studies are focused on using this finding to further improve specificity as well as elucidating the mechanistic underpinnings of low platelet contraction in ITP. Disclosures No relevant conflicts of interest to declare.
As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myri... more As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myriad of biophysical and biochemical stimuli within vasculature and heterogeneous blood clots. Highly controlled, reductionist in vitro imaging studies have been instrumental in providing a detailed and quantitative understanding of platelet biology and behavior, and have helped elucidate some surprising functions of platelets. In this review, we highlight the tools and approaches that enable visualization of platelets in conjunction with precise control over the local biofluidic and biochemical microenvironment. We also discuss next generation tools that add further control over microenvironment cell stiffness or enable visualization of the interactions between platelets and endothelial cells. Throughout the review, we include pragmatic knowledge on imaging systems, experimental conditions, and approaches that have proved to be useful to our in vitro imaging studies of platelets under flow.
Human, canine, ovine, and porcine platelets exhibit disparate biophysical signatures, whereas hum... more Human, canine, ovine, and porcine platelets exhibit disparate biophysical signatures, whereas human and murine platelets are similar. • Multiple biophysical parameters integrate during clot formation, measured by bulk clot contraction, and attenuate biophysical differences. Methods We performed a comprehensive study of platelet biophysics in humans, C57bl/6 mice, Yucatan pigs, mixed-breed hound dogs, and Hampshire cross sheep. Blood collection for this study was approved by the University of Georgia's Institutional Animal Care and Use Committee. Gel-filtered platelets were used for the adhesion assays, spreading area, and single-platelet contractile forces, as described previously. 5 (Details are provided in the supplemental Methods.) Cell adhesion and spreading assay Platelets were diluted to 20 3 10 9 /L to ensure the measurement of single platelets, not the aggregated ones; incubated on coverslips on 100 mg/mL human fibrinogen (Enzyme Research Laboratory) or type 1 rat tail collagen (VWR); and allowed to adhere for 2 hours. After imaging with bright-field microscopy, the cells were then counted and analyzed. (Details are provided in the supplemental Methods.
Health education for children with chronic illnesses (i.e., sickle cell disease [SCD]) has focuse... more Health education for children with chronic illnesses (i.e., sickle cell disease [SCD]) has focused on educating adult caregivers with minimal consideration to educating the pediatric patients. We introduce a pediatric‐focused educational paradigm, health‐related knowledge (HRK), teaching pediatric patients developmentally appropriate general health literacy, and disease‐specific knowledge. Using science, technology, engineering, and mathematics (STEM) education concepts, pediatric‐specific HRK interactive activities address educational gaps: (a) general STEM education; and (b) general health and disease‐specific knowledge to improve clinical outcomes. Total 144 pediatric SCD patients completed HRK activities, revealing overwhelmingly positive feedback (87%). Seventy‐five percent of participants in 6th grade and above demonstrated thorough understanding of the STEM/HRK topics taught.
As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myri... more As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myriad of biophysical and biochemical stimuli within vasculature and heterogeneous blood clots. Highly controlled, reductionist in vitro imaging studies have been instrumental in providing a detailed and quantitative understanding of platelet biology and behavior, and have helped elucidate some surprising functions of platelets. In this review, we highlight the tools and approaches that enable visualization of platelets in conjunction with precise control over the local biofluidic and biochemical microenvironment. We also discuss next generation tools that add further control over microenvironment cell stiffness or enable visualization of the interactions between platelets and endothelial cells. Throughout the review, we include pragmatic knowledge on imaging systems, experimental conditions, and approaches that have proved to be useful to our in vitro imaging studies of platelets under flow.
Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and ... more Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and while most of the 4,000 new cases of pediatric ITP diagnosed each year self-resolve, 10% of these patients have major bleeding episodes. The platelet count remains the mainstay method for predicting hemorrhage and unfortunately has only proven to be loosely correlated to bleeding severity. Consequently, there currently exists no biomarker that accurately and reliably predicts which patients need immediate medical treatment, all of which have side effects, and which patients only require monitoring. To that end, we leveraged our Platelet Contraction Cytometer (PCC), a versatile system that measures platelet contraction forces at the single-cell level and at high-throughput, to study platelets of patients with ITP prospectively. Buchanan bleeding scores were used to distinguish patients with severe symptoms from asymptomatic patients. With 49 patients, we observed two significant findings:...
Background: Animal models of hemostasis play a crucial role in understanding hemostasis and devel... more Background: Animal models of hemostasis play a crucial role in understanding hemostasis and developing novel therapeutic treatments. Hemostasis is a mechanical phenomenon and recent studies have demonstrated that biophysical parameters, such as platelet margination (Walton et al. Blood 2017), thrombus porosity (Welsh et al. Blood 2014), shear forces (Nesbitt et al. Nat Med 2009), compression forces (Ju et al. Nat Comm 2018), and single platelet forces, as measured by platelet contraction cytometry, developed by our laboratory, (Myers et al. Nat Mat. 2017) affect hemostatic processes and may be pathologically altered in disease states. However, little is known about the most basic biophysical interspecies differences between platelets of various animal hemostasis models, which could provide critical insight into interpreting data and results. Here, we report the results of our initial study on platelet biophysics in humans (n=5), mice (n=8), pigs (n=5), and dogs (n=6), which are comm...
Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absen... more Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absence of any known causes and affects over 4,000 US children and 8,000 adults each year (Terrell et al, 2010). Deciding when and how to treat these patients remains difficult as there is no biomarker or diagnostic test that identifies which patients will self-resolve and which are at risk for major bleeding and life-threatening intracranial hemorrhage (~10%). In addition, the medications used to treat ITP all have significant side effects, forcing clinicians to balance risks associated with bleeding and medication. As such, an ongoing debate in the field of clinical hematology centers around which patients require therapy (Flores & Buchanan, 2013)(Cooper 2017)(Gralnek 2008). Here, we show that a new quantitative measurement of platelet function, which is independent of known platelet biomarkers (Myers et al, 2017), has diagnostic potential in identifying bleeding risk in ITP patients. Speci...
Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and ... more Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and while most of the 4,000 new cases of pediatric ITP diagnosed each year self-resolve, 10% of these patients have major bleeding episodes. The platelet count remains the mainstay method for predicting hemorrhage and unfortunately has only proven to be loosely correlated to bleeding severity. Consequently, there currently exists no biomarker that accurately and reliably predicts which patients need immediate medical treatment, all of which have side effects, and which patients only require monitoring. To that end, we leveraged our Platelet Contraction Cytometer (PCC), a versatile system that measures platelet contraction forces at the single-cell level and at high-throughput, to study platelets of patients with ITP prospectively. Buchanan bleeding scores were used to distinguish patients with severe symptoms from asymptomatic patients. With 49 patients, we observed two significant findings: 1) Tracking both single platelet force measurements and platelet count enables stratifying patients into having major, minor, or no risk for bleeding. Accordingly, patients in the major risk category have a combined low platelet force and low platelet count. 2) Longitudinal studies showed that when major risk patients had increased platelet force or higher platelet counts, this was associated with the alleviation of major symptoms. Thus, when utilized together, platelet force and platelet count can more accurately predict bleeding severity in pediatric ITP patients. Platelet Contraction Cytometry: Our PCC utilizes a large array of fibrinogen "microdot" pairs patterned on a flat hydrogel of known stiffness. Thrombin-activated platelets adhere to these microdots, spread to the neighboring microdot, and contract, pulling the microdots closer together. As platelet force is directly proportional to the microdot displacement, only a single microscopy measurement is necessary to determine the force applied by each single platelet (Fig 1). As such, hundreds of platelets are measured in a controlled mechanical and biochemical environment (Myers et al, 2017) Results: With our cohort of 49 ITP patients, we observed that low average single cell platelet contraction forces highly correlate with severe bleeding symptoms (Buchannan scores 2-4). Using a regression tree, we found that an average force of 26nN best separates symptomatic from asymptomatic patients with a diagnostic sensitivity of 100% and specificity of 86.8% (AUC 0.97, 95% CI: 0.9361-1). However, platelets from some asymptomatic patients exerted forces of less than 26nN as well. In a more thorough examination, we found that majority of those asymptomatic patients with low platelet forces had platelet counts > 40,000/uL. Although our data shows that platelet count alone is a poor predictor of severe bleeding symptoms (AUC 0.8, 95% CI: 0.6757-0.9189), when a platelet count <40k/uL is coupled alongside our criterion of average contraction force <26 nN, we find that the specificity of our system increases to 94.7% and thus platelet count and platelet force synergistically predict which patients are symptomatic with high accuracy. Using a logistic regression model, we also found that with each 1nN decrease in average contraction force, the odds of the patient bleeding increases 2.1 (95% CI: 1.1-4.0) times that of being asymptomatic and when this decrease in contraction force is increased 5nN, the odds of a patient bleeding is 44.4 (95% CI: 1.8-1066.5) times that of being asymptomatic (Fig 2A-B). Moreover, when tracking individual patients (n=4) over time, we demonstrate that whenever a patient's blood sample is associated with increased single platelet average contraction force >26 nN or platelet count >40k/uL, bleeding symptoms are alleviated. Conversely, whenever a patient exhibited high platelet contractile force and low platelet count, the onset of bleeding symptoms correlated with a decrease in platelet forces. Unlike bulk or micro-clot assays, our assay functions as a "stress test" of single platelets by placing them in conditions that maximize contractility, and in both cases, the reduction in the subpopulation of highly contractile platelets is correlated with bleeding severity. As such, our work suggests that single platelet forces could be used as a diagnostic biomarker to assess bleeding risk in patients with ITP regardless of count (Fig 2C-D). Disclosures Bennett: Novartis: Research Funding. Lam:Sanguina, LLC: Equity Ownership.
One fifth of school aged children in the US have a chronic medical illness and represent an under... more One fifth of school aged children in the US have a chronic medical illness and represent an underserved and educationally disadvantaged population. In particular, children with sickle cell disease (SCD) of all genotypes spend significant amounts of time in the medical settings and represent a potentially "captive audience" where hospitalization time can include educational activities. Accordingly, we saw an opportunity for Biomedical Engineering (BME) undergraduates at Georgia Institute of Technology, who are taught biology, medicine, engineering, and design to implement a new paradigm of K-12 Science, Technology, Engineering, and Mathematics (STEM) programming, where human physiology is used as a model framework. In collaboration with hospital school teachers at Children's Healthcare of Atlanta, we created a novel educational program where we (1) leverage the patient's own medical experience as motivation for learning, (2) develop hands-on interactive activities to teach STEM concepts that are adaptable for the patient's cognitive level, (3) provide undergraduate students with high quality meaningful patient interactions and clinical experiences, and (4) provide innovative educational programming for hospitals. The BME undergraduates enrolled in a human-centered design course focusing on iterative design, development and implementation of hands-on interactive STEM activities rooted in human physiology (Fig. 1A). Each activity contains state and national K-12 STEM standards. The BME undergraduates are well-suited to teach in the hospital environment, as they experience a strong STEM curriculum and are able to capture the true interdisciplinary nature of medical science in each activity. The undergraduates and patients are near-peer age, relationship-building occurs quickly, the patients admire them and enjoy their time together. During the 2018-19 school year we developed learning assessment questions for 2 of the most requested activities, assessing each SCD patient's mastery of the learning objectives. In the Blood Jar activity (Fig. 1B), the SCD patient builds a model of blood, learning the composition, function of each component, and importance of hydration. Sixty-five patients participated, 43% of grade K - 2, 61% of 3 - 5, 85% of 6th through High School scored Thoroughly Demonstrated. In the Bone activity (Fig. 1C), each SCD patient constructs a bone model to understand the function and structure of a bone. Twenty-nine patients participated, 28% of grade K - 2, 30% of 3 - 5, 88% of 6 - 8, and 75% of high school scored Thoroughly Demonstrated. Patient feedback (Fig. 1D) has been overwhelmingly positive, stating the activities are "fun" and "engaging" and requesting additional visits from the BME undergraduates. BME undergraduate expressed growth and learning of: disease pathophysiology, teaching and learning styles, and effective and succinct communication skills. In Georgia, Life Science is taught in 5th and 7th grade, our target demographic. Our results show at least 75% of patients in 6th grade and above demonstrated thorough understanding of the STEM topics taught, while K - 5 grade did not achieve that level of understanding. Lower scores for those patients are attributed to difficulties remembering new scientific vocabulary. Many of the BME undergraduates in our program are pre-med and use the experiences and meaningful interactions with patients in their medical school applications. As stated by a BME undergraduate after a patient interaction, "the white blood cells are like soldiers in the army! I like that! This analogy prompted the patient to think of his future. It reminded me the littlest things can make a big difference. This makes me even more excited to go into medicine." Our program represents an innovative approach to teaching STEM by: engaging SCD children, who are uniquely suited to learn important STEM concepts within the context of their own disease, provide learning to SCD patients during extended absences from school, allowing BME undergraduate students to design, develop, and teach STEM activities, provide meaningful clinical experience, and supplying quality educational programming for the hospital. This collaborative program can be straightforwardly implemented at other pediatric institutions with robust college volunteer programs and hospital school programs. Our activities can be exported and implemented at other institutions. Disclosures Lam: Sanguina, Inc: Current equity holder in private company.
Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and ... more Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and while most of the 4,000 new cases of pediatric ITP diagnosed each year self-resolve, 10% of these patients have major bleeding episodes. The platelet count remains the mainstay method for predicting hemorrhage and unfortunately has only proven to be loosely correlated to bleeding severity. Consequently, there currently exists no biomarker that accurately and reliably predicts which patients need immediate medical treatment, all of which have side effects, and which patients only require monitoring. To that end, we leveraged our Platelet Contraction Cytometer (PCC), a versatile system that measures platelet contraction forces at the single-cell level and at high-throughput, to study platelets of patients with ITP prospectively. Buchanan bleeding scores were used to distinguish patients with severe symptoms from asymptomatic patients. With 49 patients, we observed two significant findings: 1) Tracking both single platelet force measurements and platelet count enables stratifying patients into having major, minor, or no risk for bleeding. Accordingly, patients in the major risk category have a combined low platelet force and low platelet count. 2) Longitudinal studies showed that when major risk patients had increased platelet force or higher platelet counts, this was associated with the alleviation of major symptoms. Thus, when utilized together, platelet force and platelet count can more accurately predict bleeding severity in pediatric ITP patients. Platelet Contraction Cytometry: Our PCC utilizes a large array of fibrinogen "microdot" pairs patterned on a flat hydrogel of known stiffness. Thrombin-activated platelets adhere to these microdots, spread to the neighboring microdot, and contract, pulling the microdots closer together. As platelet force is directly proportional to the microdot displacement, only a single microscopy measurement is necessary to determine the force applied by each single platelet (Fig 1). As such, hundreds of platelets are measured in a controlled mechanical and biochemical environment (Myers et al, 2017) Results: With our cohort of 49 ITP patients, we observed that low average single cell platelet contraction forces highly correlate with severe bleeding symptoms (Buchannan scores 2-4). Using a regression tree, we found that an average force of 26nN best separates symptomatic from asymptomatic patients with a diagnostic sensitivity of 100% and specificity of 86.8% (AUC 0.97, 95% CI: 0.9361-1). However, platelets from some asymptomatic patients exerted forces of less than 26nN as well. In a more thorough examination, we found that majority of those asymptomatic patients with low platelet forces had platelet counts > 40,000/uL. Although our data shows that platelet count alone is a poor predictor of severe bleeding symptoms (AUC 0.8, 95% CI: 0.6757-0.9189), when a platelet count <40k/uL is coupled alongside our criterion of average contraction force <26 nN, we find that the specificity of our system increases to 94.7% and thus platelet count and platelet force synergistically predict which patients are symptomatic with high accuracy. Using a logistic regression model, we also found that with each 1nN decrease in average contraction force, the odds of the patient bleeding increases 2.1 (95% CI: 1.1-4.0) times that of being asymptomatic and when this decrease in contraction force is increased 5nN, the odds of a patient bleeding is 44.4 (95% CI: 1.8-1066.5) times that of being asymptomatic (Fig 2A-B). Moreover, when tracking individual patients (n=4) over time, we demonstrate that whenever a patient's blood sample is associated with increased single platelet average contraction force >26 nN or platelet count >40k/uL, bleeding symptoms are alleviated. Conversely, whenever a patient exhibited high platelet contractile force and low platelet count, the onset of bleeding symptoms correlated with a decrease in platelet forces. Unlike bulk or micro-clot assays, our assay functions as a "stress test" of single platelets by placing them in conditions that maximize contractility, and in both cases, the reduction in the subpopulation of highly contractile platelets is correlated with bleeding severity. As such, our work suggests that single platelet forces could be used as a diagnostic biomarker to assess bleeding risk in patients with ITP regardless of count (Fig 2C-D). Disclosures Bennett: Novartis: Research Funding. Lam:Sanguina, LLC: Equity Ownership.
One fifth of school aged children in the US have a chronic medical illness and represent an under... more One fifth of school aged children in the US have a chronic medical illness and represent an underserved and educationally disadvantaged population. In particular, children with sickle cell disease (SCD) of all genotypes spend significant amounts of time in the medical settings and represent a potentially "captive audience" where hospitalization time can include educational activities. Accordingly, we saw an opportunity for Biomedical Engineering (BME) undergraduates at Georgia Institute of Technology, who are taught biology, medicine, engineering, and design to implement a new paradigm of K-12 Science, Technology, Engineering, and Mathematics (STEM) programming, where human physiology is used as a model framework. In collaboration with hospital school teachers at Children's Healthcare of Atlanta, we created a novel educational program where we (1) leverage the patient's own medical experience as motivation for learning, (2) develop hands-on interactive activities to teach STEM concepts that are adaptable for the patient's cognitive level, (3) provide undergraduate students with high quality meaningful patient interactions and clinical experiences, and (4) provide innovative educational programming for hospitals. The BME undergraduates enrolled in a human-centered design course focusing on iterative design, development and implementation of hands-on interactive STEM activities rooted in human physiology (Fig. 1A). Each activity contains state and national K-12 STEM standards. The BME undergraduates are well-suited to teach in the hospital environment, as they experience a strong STEM curriculum and are able to capture the true interdisciplinary nature of medical science in each activity. The undergraduates and patients are near-peer age, relationship-building occurs quickly, the patients admire them and enjoy their time together. During the 2018-19 school year we developed learning assessment questions for 2 of the most requested activities, assessing each SCD patient's mastery of the learning objectives. In the Blood Jar activity (Fig. 1B), the SCD patient builds a model of blood, learning the composition, function of each component, and importance of hydration. Sixty-five patients participated, 43% of grade K - 2, 61% of 3 - 5, 85% of 6th through High School scored Thoroughly Demonstrated. In the Bone activity (Fig. 1C), each SCD patient constructs a bone model to understand the function and structure of a bone. Twenty-nine patients participated, 28% of grade K - 2, 30% of 3 - 5, 88% of 6 - 8, and 75% of high school scored Thoroughly Demonstrated. Patient feedback (Fig. 1D) has been overwhelmingly positive, stating the activities are "fun" and "engaging" and requesting additional visits from the BME undergraduates. BME undergraduate expressed growth and learning of: disease pathophysiology, teaching and learning styles, and effective and succinct communication skills. In Georgia, Life Science is taught in 5th and 7th grade, our target demographic. Our results show at least 75% of patients in 6th grade and above demonstrated thorough understanding of the STEM topics taught, while K - 5 grade did not achieve that level of understanding. Lower scores for those patients are attributed to difficulties remembering new scientific vocabulary. Many of the BME undergraduates in our program are pre-med and use the experiences and meaningful interactions with patients in their medical school applications. As stated by a BME undergraduate after a patient interaction, "the white blood cells are like soldiers in the army! I like that! This analogy prompted the patient to think of his future. It reminded me the littlest things can make a big difference. This makes me even more excited to go into medicine." Our program represents an innovative approach to teaching STEM by: engaging SCD children, who are uniquely suited to learn important STEM concepts within the context of their own disease, provide learning to SCD patients during extended absences from school, allowing BME undergraduate students to design, develop, and teach STEM activities, provide meaningful clinical experience, and supplying quality educational programming for the hospital. This collaborative program can be straightforwardly implemented at other pediatric institutions with robust college volunteer programs and hospital school programs. Our activities can be exported and implemented at other institutions. Disclosures Lam: Sanguina, Inc: Current equity holder in private company.
Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absen... more Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absence of any known causes and affects over 4,000 US children and 8,000 adults each year (Terrell et al, 2010). Deciding when and how to treat these patients remains difficult as there is no biomarker or diagnostic test that identifies which patients will self-resolve and which are at risk for major bleeding and life-threatening intracranial hemorrhage (~10%). In addition, the medications used to treat ITP all have significant side effects, forcing clinicians to balance risks associated with bleeding and medication. As such, an ongoing debate in the field of clinical hematology centers around which patients require therapy (Flores & Buchanan, 2013)(Cooper 2017)(Gralnek 2008). Here, we show that a new quantitative measurement of platelet function, which is independent of known platelet biomarkers (Myers et al, 2017), has diagnostic potential in identifying bleeding risk in ITP patients. Specifically, using high-throughput platelet contraction cytometry (PCC) measurements of individual platelet forces (Myers et al, 2017), we found that low platelet forces strongly correlate with bleeding symptoms. Unlike existing tests of platelet function that use bulk measurements, our test operates at the single cell level and therefore does not depend on the platelet count, enabling a direct comparison of individual platelet function in health versus disease states. In the broad context of overall function, our findings of impaired force generation agree with previous research demonstrating that impaired platelet function, and not low platelet count, correlates with bleeding in ITP patients (Frelinger et al, 2015). In addition, as the first demonstration of a correlation between single cell force and disease, our novel approach may represent an entirely new class of "physics-based" diagnostics. Platelet Contraction Cytometry (PCC): Within the PCC, a single platelet attaches, spreads, and applies contractile force to a pair of fibrinogen microdots that are attached to a moveable, spring-like, surface. Since the applied platelet contractile force is directly proportional to the microdot displacement, the force is calculated from a single fluorescence image of the platelet. Using microfabrication technology, thousands of microdots are created on a single device to enable high-throughput measurements in tightly controlled mechanical, biochemical, and shear microenvironments (Fig 1). Here, gel-filtered platelets from patients are activated (1U/mL thrombin), plated on the device with a moderately stiff surface (75 kPa), and measured as described previously (Myers et al, 2017). Results: In a cohort of ITP patients (n = 27), we observed that patients with bleeding and/or bruising symptoms exhibited significantly lower average platelet contraction forces than asymptomatic patients regardless of platelet count. Using an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity. From a mechanistic perspective, our preliminary data also suggests that mean platelet volume does not correlate with platelet force or function, although further studies on a single platelet level are needed to confirm this result. While this data indicates that patients with bleeding symptoms have platelets with low forces, it is unclear whether low forces correlate with bleeding or whether the patient always has had low platelet forces that therefore render them susceptible to bleeding. Our preliminary prospective data suggests that low platelet forces correlate with the bleeding symptoms themselves and return to higher values when bleeding symptoms cease. Importantly, as our approach enables "single-cell" examinations of platelet forces, we identified different platelet subpopulations unique to ITP. Healthy individuals have a distribution of contractile forces with a single peak, while ITP patients tend to have two prominent peaks, one with a lower contractile force and one with a higher one. Surprisingly, patients with bleeding symptoms only have a low force peak, suggesting that the lack of highly contractile platelets may be a biophysical biomarker for bleeding (Fig 2). Ongoing studies are focused on using this finding to further improve specificity as well as elucidating the mechanistic underpinnings of low platelet contraction in ITP. Disclosures No relevant conflicts of interest to declare.
Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absen... more Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absence of any known causes and affects over 4,000 US children and 8,000 adults each year (Terrell et al, 2010). Deciding when and how to treat these patients remains difficult as there is no biomarker or diagnostic test that identifies which patients will self-resolve and which are at risk for major bleeding and life-threatening intracranial hemorrhage (~10%). In addition, the medications used to treat ITP all have significant side effects, forcing clinicians to balance risks associated with bleeding and medication. As such, an ongoing debate in the field of clinical hematology centers around which patients require therapy (Flores & Buchanan, 2013)(Cooper 2017)(Gralnek 2008). Here, we show that a new quantitative measurement of platelet function, which is independent of known platelet biomarkers (Myers et al, 2017), has diagnostic potential in identifying bleeding risk in ITP patients. Specifically, using high-throughput platelet contraction cytometry (PCC) measurements of individual platelet forces (Myers et al, 2017), we found that low platelet forces strongly correlate with bleeding symptoms. Unlike existing tests of platelet function that use bulk measurements, our test operates at the single cell level and therefore does not depend on the platelet count, enabling a direct comparison of individual platelet function in health versus disease states. In the broad context of overall function, our findings of impaired force generation agree with previous research demonstrating that impaired platelet function, and not low platelet count, correlates with bleeding in ITP patients (Frelinger et al, 2015). In addition, as the first demonstration of a correlation between single cell force and disease, our novel approach may represent an entirely new class of "physics-based" diagnostics. Platelet Contraction Cytometry (PCC): Within the PCC, a single platelet attaches, spreads, and applies contractile force to a pair of fibrinogen microdots that are attached to a moveable, spring-like, surface. Since the applied platelet contractile force is directly proportional to the microdot displacement, the force is calculated from a single fluorescence image of the platelet. Using microfabrication technology, thousands of microdots are created on a single device to enable high-throughput measurements in tightly controlled mechanical, biochemical, and shear microenvironments (Fig 1). Here, gel-filtered platelets from patients are activated (1U/mL thrombin), plated on the device with a moderately stiff surface (75 kPa), and measured as described previously (Myers et al, 2017). Results: In a cohort of ITP patients (n = 27), we observed that patients with bleeding and/or bruising symptoms exhibited significantly lower average platelet contraction forces than asymptomatic patients regardless of platelet count. Using an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity. From a mechanistic perspective, our preliminary data also suggests that mean platelet volume does not correlate with platelet force or function, although further studies on a single platelet level are needed to confirm this result. While this data indicates that patients with bleeding symptoms have platelets with low forces, it is unclear whether low forces correlate with bleeding or whether the patient always has had low platelet forces that therefore render them susceptible to bleeding. Our preliminary prospective data suggests that low platelet forces correlate with the bleeding symptoms themselves and return to higher values when bleeding symptoms cease. Importantly, as our approach enables "single-cell" examinations of platelet forces, we identified different platelet subpopulations unique to ITP. Healthy individuals have a distribution of contractile forces with a single peak, while ITP patients tend to have two prominent peaks, one with a lower contractile force and one with a higher one. Surprisingly, patients with bleeding symptoms only have a low force peak, suggesting that the lack of highly contractile platelets may be a biophysical biomarker for bleeding (Fig 2). Ongoing studies are focused on using this finding to further improve specificity as well as elucidating the mechanistic underpinnings of low platelet contraction in ITP. Disclosures No relevant conflicts of interest to declare.
As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myri... more As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myriad of biophysical and biochemical stimuli within vasculature and heterogeneous blood clots. Highly controlled, reductionist in vitro imaging studies have been instrumental in providing a detailed and quantitative understanding of platelet biology and behavior, and have helped elucidate some surprising functions of platelets. In this review, we highlight the tools and approaches that enable visualization of platelets in conjunction with precise control over the local biofluidic and biochemical microenvironment. We also discuss next generation tools that add further control over microenvironment cell stiffness or enable visualization of the interactions between platelets and endothelial cells. Throughout the review, we include pragmatic knowledge on imaging systems, experimental conditions, and approaches that have proved to be useful to our in vitro imaging studies of platelets under flow.
Human, canine, ovine, and porcine platelets exhibit disparate biophysical signatures, whereas hum... more Human, canine, ovine, and porcine platelets exhibit disparate biophysical signatures, whereas human and murine platelets are similar. • Multiple biophysical parameters integrate during clot formation, measured by bulk clot contraction, and attenuate biophysical differences. Methods We performed a comprehensive study of platelet biophysics in humans, C57bl/6 mice, Yucatan pigs, mixed-breed hound dogs, and Hampshire cross sheep. Blood collection for this study was approved by the University of Georgia's Institutional Animal Care and Use Committee. Gel-filtered platelets were used for the adhesion assays, spreading area, and single-platelet contractile forces, as described previously. 5 (Details are provided in the supplemental Methods.) Cell adhesion and spreading assay Platelets were diluted to 20 3 10 9 /L to ensure the measurement of single platelets, not the aggregated ones; incubated on coverslips on 100 mg/mL human fibrinogen (Enzyme Research Laboratory) or type 1 rat tail collagen (VWR); and allowed to adhere for 2 hours. After imaging with bright-field microscopy, the cells were then counted and analyzed. (Details are provided in the supplemental Methods.
Health education for children with chronic illnesses (i.e., sickle cell disease [SCD]) has focuse... more Health education for children with chronic illnesses (i.e., sickle cell disease [SCD]) has focused on educating adult caregivers with minimal consideration to educating the pediatric patients. We introduce a pediatric‐focused educational paradigm, health‐related knowledge (HRK), teaching pediatric patients developmentally appropriate general health literacy, and disease‐specific knowledge. Using science, technology, engineering, and mathematics (STEM) education concepts, pediatric‐specific HRK interactive activities address educational gaps: (a) general STEM education; and (b) general health and disease‐specific knowledge to improve clinical outcomes. Total 144 pediatric SCD patients completed HRK activities, revealing overwhelmingly positive feedback (87%). Seventy‐five percent of participants in 6th grade and above demonstrated thorough understanding of the STEM/HRK topics taught.
As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myri... more As the anucleate cells responsible for hemostasis and thrombosis, platelets are exposed to a myriad of biophysical and biochemical stimuli within vasculature and heterogeneous blood clots. Highly controlled, reductionist in vitro imaging studies have been instrumental in providing a detailed and quantitative understanding of platelet biology and behavior, and have helped elucidate some surprising functions of platelets. In this review, we highlight the tools and approaches that enable visualization of platelets in conjunction with precise control over the local biofluidic and biochemical microenvironment. We also discuss next generation tools that add further control over microenvironment cell stiffness or enable visualization of the interactions between platelets and endothelial cells. Throughout the review, we include pragmatic knowledge on imaging systems, experimental conditions, and approaches that have proved to be useful to our in vitro imaging studies of platelets under flow.
Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and ... more Background: Immune Thrombocytopenia Purpura (ITP) can result in severely low platelet counts and while most of the 4,000 new cases of pediatric ITP diagnosed each year self-resolve, 10% of these patients have major bleeding episodes. The platelet count remains the mainstay method for predicting hemorrhage and unfortunately has only proven to be loosely correlated to bleeding severity. Consequently, there currently exists no biomarker that accurately and reliably predicts which patients need immediate medical treatment, all of which have side effects, and which patients only require monitoring. To that end, we leveraged our Platelet Contraction Cytometer (PCC), a versatile system that measures platelet contraction forces at the single-cell level and at high-throughput, to study platelets of patients with ITP prospectively. Buchanan bleeding scores were used to distinguish patients with severe symptoms from asymptomatic patients. With 49 patients, we observed two significant findings:...
Background: Animal models of hemostasis play a crucial role in understanding hemostasis and devel... more Background: Animal models of hemostasis play a crucial role in understanding hemostasis and developing novel therapeutic treatments. Hemostasis is a mechanical phenomenon and recent studies have demonstrated that biophysical parameters, such as platelet margination (Walton et al. Blood 2017), thrombus porosity (Welsh et al. Blood 2014), shear forces (Nesbitt et al. Nat Med 2009), compression forces (Ju et al. Nat Comm 2018), and single platelet forces, as measured by platelet contraction cytometry, developed by our laboratory, (Myers et al. Nat Mat. 2017) affect hemostatic processes and may be pathologically altered in disease states. However, little is known about the most basic biophysical interspecies differences between platelets of various animal hemostasis models, which could provide critical insight into interpreting data and results. Here, we report the results of our initial study on platelet biophysics in humans (n=5), mice (n=8), pigs (n=5), and dogs (n=6), which are comm...
Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absen... more Background: Immune Thrombocytopenia Purpura (ITP) is defined by a low platelet count in the absence of any known causes and affects over 4,000 US children and 8,000 adults each year (Terrell et al, 2010). Deciding when and how to treat these patients remains difficult as there is no biomarker or diagnostic test that identifies which patients will self-resolve and which are at risk for major bleeding and life-threatening intracranial hemorrhage (~10%). In addition, the medications used to treat ITP all have significant side effects, forcing clinicians to balance risks associated with bleeding and medication. As such, an ongoing debate in the field of clinical hematology centers around which patients require therapy (Flores & Buchanan, 2013)(Cooper 2017)(Gralnek 2008). Here, we show that a new quantitative measurement of platelet function, which is independent of known platelet biomarkers (Myers et al, 2017), has diagnostic potential in identifying bleeding risk in ITP patients. Speci...
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